1. Field of the Invention
This invention relates to the analysis of particle samples using a particle analyzer.
2. Background
Particle analyzers enable the rapid analysis of particle samples to detect various characteristics of the sample as well as to detect characteristics of the individual particles. Some particle analyzers also include functionality to sort the particles according to one or more detected characteristics.
Particle analyzers, such as flow cytometers and hematology analyzers, are frequently used to analyze biological cell samples such as blood or tissue. In a flow cytometer, a cell sample is subjected to interrogation in an interrogation area along the flow path of the sample. Typically, cells in a sheath fluid pass through a flow cell, one by one, where they are interrogated by probes, including one or more beams of light. For example, one or more laser light sources can be positioned in the flow cell along the path of flow of the cell stream. In other flow cytometers, such as in jet-in-air flow cytometers, cells in a sheath fluid are interrogated by one or more probes outside of a flow cell. Several measurements are generated for each passing cell. As a cell passes through the interrogation area, resulting light characteristics, such as light scatter, light loss, and fluorescence, are measured by detectors. The measured light characteristics are used to generate corresponding electrical pulses for each interrogated cell. The electrical pulses are analyzed to determine parameters of the cell, such as, pulse peak, pulse width, and pulse area. A sorting flow cytometer, for example, can sort cells of different types into receptacles.
Prior to being interrogated, the cell sample can be prepared using various fluorochromes and/or reagents to mark specific cell types. Each fluorochrome and/or reagent can bind to cells of different types. As the cells pass through the interrogation area, laser light sources excite the fluorochromes and/or reagents. By increasing the number of different fluorochromes and/or reagents that can be detected, a cell sample can be analyzed for the presence of an increasing range of cell types. However, each laser light source, for example, can only excite fluorochromes within a limited wavelength range. It is thus desirable to use multiple laser light sources to enable the detection of a broader range of wavelengths and frequencies.
But, multiple laser light sources that are positioned along the flow path of the cell stream can lead to increased coincidence and spillover if the distances between the light sources are too small. Coincidence, i.e., the detection of more than one particle within a detection window, leads to aborting of affected particles from the analyzed sample. Spillover, i.e., the detection of the optical response generated by adjacent light sources, cause inefficiencies due to the need to compensate for spillover effects. Therefore, to avoid increased coincidence and spillover, the laser light sources are positioned with substantial distance between each other. By increasing the distance between the multiple laser light sources, the efficiency of the particle analyzer can be improved by reducing coincidences and spillover. Increased distance between light sources also enables the analysis of a range of particle sizes, thus increasing the utility of the particle analyzer even more. However, increasing the distance between laser light sources led to unexpectedly finding that the parameters generated for particles are often not accurate when the distances between laser light sources are increased.
Therefore, it is desired to improve the accuracy of parameters generated in particle analyzers that utilize multiple light sources.